Protein kinases I, II and M and their 32 P-labeled cAMP binding subunits will be further characterized using analytical isoelectric focusing techniques under denaturing and non-denaturing conditions. Peptide map analyses of cAMP-binding proteins from the three protein kinases will be carried out after labeling with the photoaffinity analog 8-N3 (32P) cAMP binding subunits will be (a) purified by electrophoresis in 0.1% SDS-7.5% polyacrylamide gels, (b) identified by autoradiography and excised, (c) digested with proteases, (d) subjected to electrophoresis in 0.1% SDS-17% polyacrylamide gels and (e) 32P-peptide patterns determined by autoradiography. The relationship between the two forms of PK II cAMP-binding proteins will also be probed by the mapping procedure. Catalytic subunits of proteins kinases I, II and M will be purified by eluting ionically-immobilized (DEAE-cellulose) holo-protein kinases with cAMP and subsequent affinity chromatography on ATP-Sepharose 4B. Purified catalytic subunits will be characterized kinetically and with respect to their rates and levels of reassociation with cAMP-binding proteins isolated from the three protein kinases. Binding subunits free of catalytic activity will be prepared by affinity chromatography on 8-amino-hexyl-cAMP-Sepharose 4B. Detergent-solubilized PK-M will be further purified by conventional techniques, preparative isoelectric focusing and affinity chromatography on ATP-Sepharose 4B. If a homogenous preparation of PK-M is obtained, its subunit structure, amino acid composition and conformational properties will be determined and compared to the properties exhibited by types I and II cytoplasmic protein kinases. An attempt will be made to isolate Friend cell mutants resistant to the effects of 8-Br-cAMP to determine whether a type I or type II protein kinase is responsible for growth regulation. Plasma membranes will be isolated from Friend cells at various stages of erythroid differentiation and the development of cAMP-dependent PK-M and its associated phosphate acceptor proteins will be delineated.